- What is the host species of the primary antibody?
- What do I need to know about the isotype of the primary antibody?
- Do I need an enzymatic or fluorescent detection?
- Do I need a pre-adsorbed secondary antibody?
- Do I need an affinity purified antibody or IgG fraction?
- Is it necessary to use a F(ab) or F(ab')2 fragment antibody?
- Do I need an IgG H&L, light chain or F(ab')2 against F(ab')2 secondary antibody?
The secondary antibody is directed against the species of the primary antibody. If you use a primary antibody raised in rabbit, you will need an anti-rabbit secondary antibody raised in a species other than rabbit. For example, goat polyclonal to rabbit IgG - H&L (HRP) (ab6721) can be used to detect rabbit polyclonal to Ki67 (ab15580).
The secondary antibody has to be directed against the isotype of the primary antibody.
Polyclonal primary antibodies are generally raised in rabbit, goat, sheep or donkey and are an IgG isotype. The secondary antibody will typically be an anti-IgG H&L (Heavy & Light chains) antibody.
Monoclonal primary antibodies are commonly raised in mouse, rabbit and rat. For example, if the primary monoclonal antibody is a mouse IgG1, you will need an anti-mouse IgG or a less specific F(ab) fragment anti-mouse IgG.
Human immunoglobulin classes, subclasses, types and subtypes:
- Classes or isotypes: IgG (γ heavy chains), IgM (μ), IgA (α), IgE (ε), IgD (δ)
- Subclasses: IgG1 (γ1 heavy chains), IgG2 (γ2), IgG3 (γ3), IgG4 (γ4); IgA1 (α1), IgA2 (α2)
- Types: κ light chain, λ light chain
- Subtypes: λ1, λ2, λ3, λ4
Other type of reactivities:
- Polyvalent antibodies react with all classes
- Anti-Fc or heavy chain (α, δ, ε, γ, and μ) antibodies react with heavy chain only
- Anti-F(ab) or whole molecule antibodies react with heavy and light chains independently of the class
- Anti-light chain (κ and λ) antibodies react with all classes since all classes use the same κ and λ light chains
The type of conjugation is application dependent.
For enzymatic and biotin detection, e.g. in WB or ELISA, we suggest a secondary antibody conjugated to horseradish peroxidase (HRP), alkaline phosphatase (AP) or biotin. Both avidin and streptavidin bind very strongly to biotin and enable signal amplification, regardless of the host species of the antibody.
If a laser light is involved, e.g. in Flow Cytometry, ICC/IF or IHC, we suggest fluorescent detection with a secondary antibody conjugated to a fluorochrome. Explore our guide to fluorochromes to choose a dye for your experiments.
Learn more about the characteristics and advantages of using DyLight® fluorochromes.
We usually recommend using a secondary antibody pre-adsorbed with serum, for western blotting of immunoglobulin-rich tissues and cells. Pre-adsorbed secondary antibodies are less likely to interact with endogenous immunolgobulins and consequently may reduce non-specific background. The secondary antibody should be pre-adsorbed against the same species as the sample on which the detection is performed. For example, a human pre-adsorbed antibody will be required for detection in human tissue.
Learn more about DyLight® pre-adsorbed secondary antibodies.
The advantage of using affinity purified antibodies or IgG fractions will depend on the type of binding expected. Affinity purified antibodies give the lowest amount of non-specific binding whereas IgG fractions contain high affinity antibodies. Indeed, during an affinity purification, high affinity antibodies stay fixed on the matrix and cannot be eluted.
F(ab) and F(ab')2 fragment antibodies eliminate non-specific binding between Fc portions of antibodies and Fc receptors on cells (such as macrophages, dendritic cells, neutrophils, NK cells and B cells) and penetrate tissues more efficiently due to their smaller size. As fragment antibodies do not have Fc portions, they do not interfere with anti-Fc mediated antibody detection.
Explore the advantages of F(ab) or F(ab')2 fragments.
Our secondary antibodies are supplied in different formats:
- Anti-IgG H&L antibodies react with both heavy and light chains of IgG and subclasses
- Anti-light chain antibodies react with the light chain of primary antibodies which is the same among all classes
- Anti-F(ab')2 secondary antibodies react with the F(ab')2 portion of the primary antibody
Explore the advantages of using F(ab) or F(ab')2 fragments.